Method of Using dihydro-resveratrol or its stilbenoid derivatives and/or chemical variants in treatments of fibrotic and diabetic conditions

ABSTRACT

A polyphenol derivative of the stilbenoid family, namely trans-3,5,4′-trihydroxybibenzyl, also known as dihydro-resveratrol, is disclosed as a remedial agent. In particular, the usage of dihydro-resveratrol as an anti-fibrotic agent in suppressing the activation of pancreatic stellate cells (PSCs) is presented. One embodiment also relates to the management of pancreatic fibrosis, which is often accompanied with chronic pancreatitis and desmoplastic reaction of pancreatic cancer. Another embodiment has applications in preventing or alleviating or treating pancreatic cancer or pancreatogenic diabetes.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part application of theU.S. non-provisional patent application Ser. No. 14/740,410 filed onJun. 16, 2015, which disclosure is hereby incorporated by reference inits entirety.

FIELD OF INVENTION

The present invention relates to a polyphenol derivative of thestilbenoid family, namely trans-3,5,4′-trihydroxybibenzyl, also known asdihydro-resveratrol, as a remedial agent. In particular, the presentinvention presents the usage of dihydro-resveratrol as an anti-fibroticagent in suppressing the activation of pancreatic stellate cells (PSCs).One embodiment of the present invention also relates to the managementof pancreatic fibrosis, which is often accompanied with chronicpancreatitis and desmoplastic reaction of pancreatic cancer. Anotherembodiment of the present invention also has applications in preventingor alleviating or treating chronic pancreatitis and the associateddiseases, such as pancreatic tumors and pancreatogenic diabetes.

BACKGROUND OF INVENTION

Pancreatic fibrosis is a pronounced histopathological feature of chronicpancreatitis, which is an active dynamic process resulting inirreversible tissue scarring and morphological alteration of thepancreatic parenchyma. The long-standing or recurrent inflammation ofthe pancreas is often associated with progressive fibrosis; thus, causespatients persistent abdominal pain and permanent impairment ofpancreatic functions, and eventually leads to various systemiccomplications, including malabsorption and diabetes. According to anumber of recent studies, pancreatic stellate cells (PSCs) play acritical role in the development of pancreatic fibrogenesis which isoften accompanied with chronic pancreatitis and desmoplastic reaction ofpancreatic cancer [Patel M and Fine D R 2005]. Typically, PSCs localizedin the periacinar region of the exocrine pancreas are quiescent innormal condition. Upon injury or inflammation, these PSCs tend to losetheir fat-droplets and transform into myofibroblast-like phenotype whichcan be identified with the presence of α-smooth muscle actin (α-SMA orActa2) [Apte et al 1998]. The formation of these fibrotic stressfilaments actually elicits the cascade of tissue repairing mechanisms inresponse to pro-fibrotic or pro-inflammatory mediators such astransforming growth factor-beta (TGF-β) generated against tissue injury[Apte et al 2004; Yoo B M et al 2005]. Once the PSCs are activated, theyproduce massive extracellular matrix (ECM) proteins, namely fibronectin1 (FN1) and type I collagen, for the purpose of tissue repairing as wellas regeneration [Schneider E et al 2001]. In fact, properties ofstellate cells in the pancreas are similar to those present in otherorgans such as liver, kidney and lung. The overwhelmed production of ECMin an organ causes scarring of the parenchyma and leads to permanentmorphological damages of the organ. Progressive fibrosis probablyresults in anatomical anomalies and organ failure. In the pancreas, thefibrotic condition may eventually lead to pancreatic exocrine and/orendocrine insufficiency. Therefore, the abolishment of the PSCactivation is crucial to the treatment of chronic pancreatitis,explicitly fibrogenesis and the associated impairments of pancreaticfunctions, including pancreatogenic diabetes, also known as Type 3cdiabetes milieus (T3cDM). Recent clinical data indicates that about 80%of T3cDM cases are arisen from chronic pancreatitis whereas 30-50% ofchronic pancreatitis patients develop diabetes as the disease progresses[Ewald N and Hardt P D. 2013].

It is an objective of the present invention to provide a method of usingdihydro-resveratrol as an anti-fibrotic agent in a subject.

Citation or identification of any reference in this section or any othersection of this application shall not be construed as an admission thatsuch reference is available as prior art for the present application.

SUMMARY OF INVENTION

Accordingly, the objective of the present invention relates to a methodof using an effective amount of dihydro-resveratrol or its stilbenoidderivatives and/or chemical variants as a potential remedial agent forthe treatment and/or alleviation of fibrotic conditions such aspancreatic fibrosis in chronic pancreatitis in a subject in needsthereof. Dihydro-resveratrol effectively reduces fibrotic stressfilaments α-SMA and PSC phenotype in the pancreas. Further,dihydro-resveratrol rectifies hyperglycemic condition in pancreatogenicdiabetes, which is emerged as a sequela secondary to chronicpancreatitis. Importantly, oral administration of dihydro-resveratrolshows no adverse effects in the animal trial.

In a first embodiment of a first aspect of the present invention thereis provided a method of treating acute inflammatory condition of thepancreas and associated systemic complications by administering to asubject in needs thereof a composition comprising an effective amount ofa stilbenoid derivative which comprises a compound of formula (1),

wherein R₁, R₂ and R₃ are each independently selected from an alkylgroup. The term “alkyl”, alone or in combination with other groups,includes reference to a straight chain alkyl moiety having 1, 2, 3, 4, 5or 6 carbon atoms. The term is further exemplified by such groups asmethyl, ethyl, propyl (n-propyl or isopropyl), butyl (n-butyl, sec-butyland tert-butyl), pentyl, hexyl and the like, and the derivatives orchemical variants thereof; or a mixture of said compound, the derivativeand/or chemical variants thereof.

In a second embodiment of a first aspect of the present invention thereis provided a method of treating acute inflammatory condition of thepancreas and associated systemic complications wherein the stilbenoidderivative is trans-3,5,4′-trihydroxybibenzyl, or dihydro-resveratrol,which is a compound of formula (2):

and the derivatives or chemical variants thereof; or a mixture of saidcompound, the derivative and/or chemical variants thereof.

In a third embodiment of the first aspect of the present invention thereis provided a method of treating acute inflammatory condition of thepancreas and associated systemic complications wherein the subject is ahuman or an animal.

In a fourth embodiment of the first aspect of the present inventionthere is provided a method of treating acute inflammatory condition ofthe pancreas and associated systemic complications wherein thecomposition is administered orally.

In a fifth embodiment of the first aspect of the present invention thereis provided a method of treating acute inflammatory condition of thepancreas and associated systemic complications wherein the acuteinflammatory condition of the pancreas comprises all forms of acutepancreatitis and the associated systemic complications comprisepulmonary injury.

In a sixth embodiment of the first aspect of the present invention thereis provided a method of treating acute inflammatory condition of thepancreas and associated systemic complications wherein said compositionis administered at no less than 20 mg/kg to said subject for no lessthan 3 times a day.

In a seventh embodiment of the first aspect of the present inventionthere is provided a method of treating acute inflammatory condition ofthe pancreas and associated systemic complications wherein saidcomposition is administered at no less than 3.24 mg/kg to said subjectfor no less than 3 times a day.

In a first embodiment of a second aspect of the present invention thereis provided a method for preparing a compound of molecular formulaC₁₄H₁₄O₃ and of formula (2),

which is a stilbenoid derivative having a chemical name oftrans-3,5,4′-trihydroxybibenzyl by hydrogenating of trans-resveratrol.

In a second embodiment of the second aspect of the present inventionthere is provided a method of preparing the compound of molecularformula C₁₄H₁₄O₃ and of formula (2) wherein the hydrogenating oftrans-resveratrol comprises steps of

-   -   stirring a solution of trans-resveratrol in anhydrous ethanol        (EtOH) at room temperature under 5 atm H₂ pressure in the        presence of 10% Pd/C for 8 hours;    -   filtering off the catalyst from the stirred solution;    -   evaporating the filtrate in vacuum to produce a residue;    -   eluting the residue using silica gel chromatographic separation        with petroleum ether and ethyl acetate (1:1) to produce        dihydro-resveratrol.

In a first embodiment of the third aspect of the present invention thereis provided a compound for suppressing a fibrotic mediator of stellatecells present in an internal organ of a subject in need with a formulaof

wherein

R² and R⁴ are each independently selected from —OR¹¹ and —OC(O)R¹¹;

R¹, R³, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are each independently selected fromhydrogen, halogen, trifluoromethyl, —OR¹¹ and —OC(O)R¹¹; or R² and R³,or R⁷ and R⁸ may be taken together with the carbon atoms to which theyare attached to form a cyclic group;

R¹¹ is independently hydrogen or selected from hydrocarbyl andheterocyclyl, either of which is optionally substituted with 1, 2, 3, 4or 5 R¹²;

R¹² is independently selected from halogen, trifluoromethyl, cyano,nitro, oxo, —OR¹³, —C(O)R¹⁴, —C(O)N(R¹³)R¹⁴, —C(O)OR¹³, —OC(O)R¹⁴,—S(O)₂R¹³, —S(O)₂N(R¹³)R¹⁴, —N(R¹³)R¹⁴;

R¹³ and R¹⁴ are each independently hydrogen or selected from hydrocarbyland heterocyclyl, either of which is optionally substituted with 1, 2,3, 4 or 5 substituents independently selected from halogen, cyano,amino, hydroxy, C₁₋₆ alkyl and C₁₋₆ alkoxy;

or an enantiomer thereof;

or a pharmaceutically acceptable salt or prodrug thereof;

or a mixture of said compound, the derivative and/or chemical variantsthereof.

In a second embodiment of the third aspect of the present inventionthere is provided a compound for suppressing a fibrotic mediator ofstellate cells present in an internal organ of a subject in need with aformula of

In a first embodiment of the fourth aspect of the present inventionthere is provided a method for suppressing fibrotic mediator(s) instellate cells present in an internal organ of a subject in need byusing a composition comprising compounds i to viii ordihydro-resveratrol.

In a second embodiment of the fourth aspect of the present inventionthere is provided a method for suppressing fibrotic mediator(s) instellate cells present in an internal organ of a subject in need whereinthe composition is administered to a subject in need thereof with adosage of at least 1.622 mg/kg/day wherein said composition is consistedof a compound with a formula of

In a third embodiment of the fourth aspect of the present inventionthere is provided a method for suppressing fibrotic mediator(s) instellate cells present in an internal organ of a subject in need whereinthe said subject is human.

In a fourth embodiment of the fourth aspect of the present inventionthere is provided a method for suppressing fibrotic mediator(s) instellate cells present in an internal organ of a subject in need whereinthe composition is administered orally to said subject in need thereof.

In a fifth embodiment of the fourth aspect of the present inventionthere is provided a method for suppressing fibrotic mediator(s) instellate cells present in an internal organ of a subject in need whereinthe internal organ comprises pancreas, liver, kidney and lung.

In a first embodiment of the fifth aspect of the present invention thereis provided a compound for treating pancreatogenic diabetes (or Type 3cdiabetes milieus) in a subject in need with a therapeutically effectiveamount of a formula of

wherein

R² and R⁴ are each independently selected from —OR¹¹ and —OC(O)R¹¹;

R¹, R³, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are each independently selected fromhydrogen, halogen, trifluoromethyl, —OR¹¹ and —OC(O)R¹¹; or R² and R³,or R⁷ and R⁸ may be taken together with the carbon atoms to which theyare attached to form a cyclic group;

R¹¹ is independently hydrogen or selected from hydrocarbyl andheterocyclyl, either of which is optionally substituted with 1, 2, 3, 4or 5 R¹²;

R¹² is independently selected from halogen, trifluoromethyl, cyano,nitro, oxo, —OR¹³, —C(O)R¹⁴, —C(O)N(R¹³)R¹⁴, —C(O)OR¹³, —OC(O)R¹⁴,—S(O)₂R¹³, —S(O)₂N(R¹³)R¹⁴, —N(R¹³)R¹⁴;

R¹³ and R¹⁴ are each independently hydrogen or selected from hydrocarbyland heterocyclyl, either of which is optionally substituted with 1, 2,3, 4 or 5 substituents independently selected from halogen, cyano,amino, hydroxy, C₁₋₆ alkyl and C₁₋₆ alkoxy;

or an enantiomer thereof;

or a pharmaceutically acceptable salt or prodrug thereof;

or a mixture of said compound, the derivative and/or chemical variantsthereof.

In a second embodiment of the fifth aspect of the present inventionthere is provided a method for treating pancreatogenic diabetes (or Type3c diabetes milieus) in a subject in need by using a compositioncomprising compounds of a formula of

wherein

R² and R⁴ are each independently selected from —OR¹¹ and —OC(O)R¹¹;

R¹, R³, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are each independently selected fromhydrogen, halogen, trifluoromethyl, —OR¹¹ and —OC(O)R¹¹; or R² and R³,or R⁷ and R⁸ may be taken together with the carbon atoms to which theyare attached to form a cyclic group;

R¹¹ is independently hydrogen or selected from hydrocarbyl andheterocyclyl, either of which is optionally substituted with 1, 2, 3, 4or 5 R¹²;

R¹² is independently selected from halogen, trifluoromethyl, cyano,nitro, oxo, —OR¹³, —C(O)R¹⁴, —C(O)N(R¹³)R¹⁴, —C(O)OR¹³, —OC(O)R¹⁴,—S(O)₂R¹³, —S(O)₂N(R¹³)R¹⁴, —N(R¹³)R¹⁴

R¹³ and R¹⁴ are each independently hydrogen or selected from hydrocarbyland heterocyclyl, either of which is optionally substituted with 1, 2,3, 4 or 5 substituents independently selected from halogen, cyano,amino, hydroxy, C₁₋₆ alkyl and C₁₋₆ alkoxy;

or an enantiomer thereof;

or a pharmaceutically acceptable salt or prodrug thereof;

or a mixture of said compound, the derivative and/or chemical variantsthereof.

In a third embodiment of the fifth aspect of the present invention thereis provided a method for treating pancreatogenic diabetes (or Type 3cdiabetes milieus) in a subject in need wherein the composition isadministered to a subject in need thereof with a dosage of at least1.622 mg/kg/day wherein said composition is consist of a compound with aformula of

In a fourth embodiment of the fifth aspect of the present inventionthere is provided a method for treating pancreatogenic diabetes (or Type3c diabetes milieus) in a subject in need wherein the said subject ishuman.

In a fifth embodiment of the fifth aspect of the present invention thereis provided a method for treating pancreatogenic diabetes (or Type 3cdiabetes milieus) in a subject in need wherein the composition isadministered orally to said subject in need thereof.

In a first embodiment of the sixth aspect of the present invention thereis provided a compound for treating pancreatogenic diabetes (or Type 3cdiabetes milieus) in a subject in need with a therapeutically effectiveamount of a formula of

In a second embodiment of the sixth aspect of the present inventionthere is provided a method for treating pancreatogenic diabetes (orType3c diabetes milieus) in a subject wherein the composition isadministered to a subject in need thereof with a dosage of at least1.622 mg/kg/day wherein said composition is consist of a compound with aformula of

In a third embodiment of the sixth aspect of the present invention thereis provided a method for treating pancreatogenic diabetes (or Type3cdiabetes milieus) in a subject wherein the said subject is human.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described.

The invention includes all such variation and modifications. Theinvention also includes all of the steps and features referred to orindicated in the specification, individually or collectively and any andall combinations or any two or more of the steps or features.

Throughout this specification, unless the context requires otherwise,the word “comprise” or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated integer or groupof integers but not the exclusion of any other integer or group ofintegers. It is also noted that in this disclosure and particularly inthe claims and/or paragraphs, terms such as “comprises”, “comprised”,“comprising” and the like can have the meaning attributed to it in U.S.Patent law; e.g., they can mean “includes”, “included”, “including”, andthe like; and that terms such as “consisting essentially of” and“consists essentially of” have the meaning ascribed to them in U.S.Patent law, e.g., they allow for elements not explicitly recited, butexclude elements that are found in the prior art or that affect a basicor novel characteristic of the invention.

Furthermore, throughout the specification and claims, unless the contextrequires otherwise, the word “include” or variations such as “includes”or “including”, will be understood to imply the inclusion of a statedinteger or group of integers but not the exclusion of any other integeror group of integers.

Other definitions for selected terms used herein may be found within thedetailed description of the invention and apply throughout. Unlessotherwise defined, all other technical terms used herein have the samemeaning as commonly understood to one of ordinary skill in the art towhich the invention belongs.

Other aspects and advantages of the invention will be apparent to thoseskilled in the art from a review of the ensuing description.

BRIEF DESCRIPTION OF DRAWINGS

The above and other objects and features of the present invention willbecome apparent from the following description of the present invention,when taken in conjunction with the accompanying drawings, in which:

FIG. 1 shows the gain of water content in rats due to effect ofpancreatic edema as a result of cerulein-induced acute pancreatitis. Theobtained weights are expressed as a ratio percentage of pancreaticweight to body mass. A p-value of less than 0.05 is considered asstatistically significant. *p<0.05 when comparing with control groupwhereas #p<0.05 comparing with cerulein group.

FIGS. 2A to 2F show the architecture and morphological alteration inpancreatic tissues of control group (FIG. 2A), cerulein group (FIG. 2B)and dihydro-resveratrol treatment groups (D-Res) (FIGS. 2C to 2F) bymeans of hematoxylin and eosin (H&E) staining. Images are shown with ascale bar of 50 m.

FIGS. 3A to 3E show the architecture and morphological alteration inpulmonary tissues of control group (FIG. 3A), cerulein group (FIG. 3B)and dihydro-resveratrol treatment groups (D-Res) (FIGS. 3C to 3E) bymeans of H&E staining. Images are shown with a magnification of 200×.

FIG. 4A shows the measurement of MPO activity which representsneutrophil sequestration in pancreatic tissues of control group,cerulein group and dihydro-resveratrol treatment groups (D-Res) by meansof colorimetric spectrophotometry. A p-value of less than 0.05 isconsidered as statistically significant. *p<0.05 when comparing withcontrol group whereas #p<0.05 comparing with cerulein group.

FIG. 4B shows the measurement of MPO activity which representsneutrophil sequestration in pulmonary tissues of control group, ceruleingroup and dihydro-resveratrol treatment groups (D-Res) by means ofcolorimetric spectrophotometry. A p-value of less than 0.05 isconsidered as statistically significant. *p<0.05 when comparing withcontrol group whereas #p<0.05 comparing with cerulein group.

FIG. 5A shows the measurement of TNF-α level in pancreatic tissues ofcontrol group, cerulein group and dihydro-resveratrol treatment groups(D-Res) by means of enzyme-linked immunosorbent assay (ELISA). A p-valueof less than 0.05 is considered as statistically significant. *p<0.05when comparing with control group whereas #p<0.05 comparing withcerulein group.

FIG. 5B shows the measurement of TNF-α level in pulmonary tissues ofcontrol group, cerulein group and dihydro-resveratrol treatment groups(D-Res) by means of ELISA. A p-value of less than 0.05 is considered asstatistically significant. *p<0.05 when comparing with control groupwhereas #p<0.05 comparing with cerulein group.

FIG. 6A shows the measurement of glutathione levels in pancreatictissues of control group, cerulein group and dihydro-resveratroltreatment groups (D-Res) by means of colorimetric spectrophotometry. Ap-value of less than 0.05 is considered as statistically significant.*p<0.05 when comparing with control group whereas #p<0.05 comparing withcerulein group.

FIG. 6B shows the ameliorative effect of dihydro-resveratrol (D-res) andtrans-resveratrol (Res) on reducing water content as a result ofpancreatic edema in rats with cerulein-induced acute pancreatitis. Theobtained weights are expressed as a ratio percentage of pancreaticweight to body mass. A p-value of less than 0.05 is considered asstatistically significant. *p<0.05 when comparing with saline-treatedcontrol group (Con) whereas #p<0.05 comparing with cerulein-treatedcontrol group.

FIG. 7 shows the measurement of metabolic rates by means of MTT cellproliferation in pancreatic acinar cells treated withdihydro-resveratrol and trans-resveratrol. *p<0.05 when comparing withcells treated without dihydro-resveratrol or trans-resveratrol.

FIG. 8A to 8H shows eight derivatives (i.e. Compound i to Compound viii)from dihydro-resveratrol (i.e. Compound 2).

FIG. 81 shows the chemical structure of dihydro-resveratrol (i.e.Compound 2).

FIG. 9 shows Western blotting of LTC-14 PSCs pre-incubated with TGF-β (5ng/mL), and treated with trans-resveratrol (Resv) or dihydro-resveratrolor stilbene compounds i to viii at 20 μg/mL for 24 hours. Control wasnot treated with Resv or any stilbenoids.

FIG. 10 shows the probing of α-SMA and FN1 in Western blotting of LTC-14cells pre-incubated with TGF-β (5 ng/mL), and treated withtrans-resveratrol or dihydro-resveratrol at the indicatedconcentrations.

FIG. 11 shows the green fluorescent signal (identified by arrow marks)of fibrotic filaments α-SMA in LTC-14 cells implying the degree of PSCactivation.

FIG. 12 shows the fluorescent signal of fibronectin (FN1) deposition inpancreatic tissue sections for an estimation of the degree of fibrosiswith and without treatment of dihydro-resveratrol (D-Res, 20 mg/kg/day)or trans-resveratrol (Res, 20 mg/kg/day) in cerulein (Cer)-inducedchronic pancreatitis mice.

FIG. 13 shows the glucose response of the normal mice (Control) andchronic pancreatitis mice (Cer) with or without treatment ofdihydro-resveratrol (D-Res, 20 mg/kg/day) in the intraperitoneal glucosetolerance test. At all time-points, a significant difference (p<0.05) isachieved between the Cer group and the Cer+D-Res group.

FIG. 14 shows the fluorescent signal of insulin in pancreatic tissuesections for an evaluation of pancreatic insulin-secreting cell (i.e.beta-cell) area. The comparison is made among the control mice, chronicpancreatitis mice and the chronic pancreatitis mice with treatment ofdihydro-resveratrol (D-Res) at 20 mg/kg/day.

DETAILED DESCRIPTION OF INVENTION

The present invention is not to be limited in scope by any of thespecific embodiments described herein. The following embodiments arepresented for exemplification only.

DEFINITIONS Hydrocarbyl

The term “hydrocarbyl” as used herein includes reference to a moietyconsisting exclusively of hydrogen and carbon atoms; such a moiety maycomprise an aliphatic and/or an aromatic moiety. The moiety may comprise1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20carbon atoms. Examples of hydrocarbyl groups include C₁₋₆ alkyl (e.g.C₁, C₂, C₃ or C₄ alkyl, for example methyl, ethyl, propyl, isopropyl,n-butyl, sec-butyl or tert-butyl); C₁₋₆ alkyl substituted by aryl (e.g.benzyl) or by cycloalkyl (e.g. cyclopropylmethyl); cycloalkyl (e.g.cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl); aryl (e.g. phenyl,naphthyl or fluorenyl) and the like.

Alkyl

The term “alkyl” as used herein includes reference to a straight orbranched chain alkyl moiety having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. Examples of alkylgroups include “C₁₋₆ alkyl” and “C₂₋₁₀ alkyl”. The term “C₁₋₆ alkyl” asused herein include reference to a straight or branched chain alkylmoiety having 1, 2, 3, 4, 5 or 6 carbon atoms. The term “C₂₋₁₀ alkyl” asused herein include reference to a straight or branched chain alkylmoiety having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. This termincludes reference to groups such as methyl, ethyl, propyl (n-propyl orisopropyl), butyl (n-butyl, sec-butyl or tert-butyl), pentyl, hexyl andthe like. In particular, the alkyl moiety may have 1, 2, 3, 4, 5 or 6carbon atoms.

Alkenyl

The terms “alkenyl” and “C₂₋₆ alkenyl” as used herein include referenceto a straight or branched chain alkyl moiety having 2, 3, 4, 5 or 6carbon atoms and having, in addition, at least one double bond, ofeither E or Z stereochemistry where applicable. This term includesreference to groups such as ethenyl, 2-propenyl, 1-butenyl, 2-butenyl,3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1-hexenyl, 2-hexenyl and3-hexenyl and the like.

Alkynyl

The terms “alkynyl” and “C₂₋₆ alkynyl” as used herein include referenceto a straight or branched chain alkyl moiety having 2, 3, 4, 5 or 6carbon atoms and having, in addition, at least one triple bond. Thisterm includes reference to groups such as ethynyl, 1-propynyl,2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl,3-pentynyl, 1-hexynyl, 2-hexynyl and 3-hexynyl and the like.

Alkoxy

The terms “alkoxy” and “C₁₋₆ alkoxy” as used herein include reference to—O-alkyl, wherein alkyl is straight or branched chain and comprises 1,2, 3, 4, 5 or 6 carbon atoms. In one class of embodiments, alkoxy has 1,2, 3 or 4 carbon atoms. This term includes reference to groups such asmethoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy, pentoxy,hexoxy and the like.

Cycloalkyl

The term “cycloalkyl” as used herein includes reference to an alicyclicmoiety having 3, 4, 5, 6, 7 or 8 carbon atoms. The group may be abridged or polycyclic ring system. More often cycloalkyl groups aremonocyclic. This term includes reference to groups such as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, bicyclo[2.2.2]octyl andthe like.

Aryl

The term “aryl” as used herein includes reference to an aromatic ringsystem comprising 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring carbonatoms. Aryl is often phenyl but may be a polycyclic ring system, havingtwo or more rings, at least one of which is aromatic. This term includesreference to groups such as phenyl, naphthyl, fluorenyl, azulenyl,indenyl, anthryl and the like.

Cyclic Group

“Cyclic group” means a ring or ring system, which may be unsaturated orpartially unsaturated but is usually saturated, typically containing 5to 13 ring-forming atoms, for example a 5- or 6-membered ring. The ringor ring system may be substituted with one or more hydrocarbyl groups.Cyclic group includes carbocyclyl and heterocyclyl moeities.

Carbocyclyl

The term “carbocyclyl” as used herein includes reference to a saturated(e.g. cycloalkyl) or unsaturated (e.g. aryl) ring moiety having 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 carbon ring atoms. Inparticular, carbocyclyl includes a 3- to 10-membered ring or ring systemand, in particular, 5- or 6-membered rings, which may be saturated orunsaturated. The ring or ring system may be substituted with one or morehydrocarbyl groups. A carbocyclic moiety is, for example, selected fromcyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl,bicyclo[2.2.2]octyl, phenyl, naphthyl, fluorenyl, azulenyl, indenyl,anthryl and the like.

Heterocyclyl

The term “heterocyclyl” as used herein includes reference to a saturated(e.g. heterocycloalkyl) or unsaturated (e.g. heteroaryl) heterocyclicring moiety having from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or16 ring atoms, at least one of which is selected from nitrogen, oxygen,phosphorus, silicon and sulphur. In particular, heterocyclyl includes a3- to 10-membered ring or ring system and more particularly a 5- or6-membered ring, which may be saturated or unsaturated. The ring or ringsystem may be substituted with one or more hydrocarbyl groups.

A heterocyclic moiety is, for example, selected from oxiranyl, azirinyl,1, 2-oxathiolanyl, imidazolyl, thienyl, furyl, tetrahydrofuryl, pyranyl,thiopyranyl, thianthrenyl, isobenzofuranyl, benzofuranyl, chromenyl,2H-pyrrolyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrrolizidinyl,imidazolyl, imidazolidinyl, benzimidazolyl, pyrazolyl, pyrazinyl,pyrazolidinyl, thiazolyl, isothiazolyl, dithiazolyl, oxazolyl,isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, piperidyl, piperazinyl,pyridazinyl, morpholinyl, thiomorpholinyl, especially thiomorpholino,indolizinyl, isoindolyl, 3H-indolyl, indolyl, benzimidazolyl, cumaryl,indazolyl, triazolyl, tetrazolyl, purinyl, 4/V-quinolizinyl,isoquinolyl, quinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl,decahydroquinolyl, octahydroisoquinolyl, benzofuranyl, dibenzofuranyl,benzothiophenyl, dibenzothiophenyl, phthalazinyl, naphthyridinyl,quinoxalyl, quinazolinyl, quinazolinyl, cinnolinyl, pteridinyl,carbazoiyl, β-carbolinyl, phenanthridinyl, acridinyl, perimidinyl,phenanthrolinyl, furazanyl, phenazinyl, phenothiazinyl, phenoxazinyl,chromenyl, isochromanyl, chromanyl and the like.

Heterocycloalkyl

The term “heterocycloalkyl” as used herein includes reference to asaturated heterocyclic moiety having 3, 4, 5, 6 or 7 ring carbon atomsand 1, 2, 3, 4 or 5 ring heteroatoms selected from nitrogen, oxygen,phosphorus and sulphur. The group may be a polycyclic ring system butmore often is monocyclic. This term includes reference to groups such asazetidinyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, oxiranyl,pyrazolidinyl, imidazolyl, indolizidinyl, piperazinyl, thiazolidinyl,morpholinyl, thiomorpholinyl, quinolizidinyl and the like. The ring orring system may be substituted with one or more hydrocarbyl groups.

Heteroaryl

The term “heteroaryl” as used herein includes reference to an aromaticheterocyclic ring system having 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or16 ring atoms, at least one of which is selected from nitrogen, oxygenand sulphur. The group may be a polycyclic ring system, having two ormore rings, at least one of which is aromatic, but is more oftenmonocyclic. The ring or ring system may be substituted with one or morehydrocarbyl groups. This term includes reference to groups such aspyrimidinyl, furanyl, benzo[b]thiophenyl, thiophenyl, pyrrolyl,imidazolyl, pyrrolidinyl, pyridinyl, benzo[b]furanyl, pyrazinyl,purinyl, indolyl, benzimidazolyl, quinolinyl, phenothiazinyl, triazinyl,phthalazinyl, 2H-chromenyl, oxazolyl, isoxazolyl, thiazolyl, isoindolyl,indazolyl, purinyl, isoquinolinyl, quinazolinyl, pteridinyl and thelike.

Halogen

The term “halogen” as used herein includes reference to F, Cl, Br or I.

Halogen Containing Moiety

The expression “halogen containing moiety” as used herein includesreference to a moiety comprising 1 to 30 plural valence atoms selectedfrom carbon, nitrogen, oxygen and sulphur which moiety includes at leastone halogen. The moiety may be hydrocarbyl for example C₁₋₆ alkyl orC₁₋₆ alkoxy, or carbocyclyl for example aryl.

Substituted

The term “substituted” as used herein in reference to a moiety meansthat one or more, especially up to 5, more especially 1, 2 or 3, of thehydrogen atoms in said moiety are replaced independently of each otherby the corresponding number of the described substituents. The term“optionally substituted” as used herein means substituted orun-substituted. It will, of course, be understood that substituents areonly at positions where they are chemically possible, the person skilledin the art being able to decide (either experimentally or theoretically)without inappropriate effort whether a particular substitution ispossible.

Enantiomer

The term “enantiomer” as used herein means one of two stereoisomers thathave mirror images of one another.

Racemate

The term “racemate” as used herein means a mixture of equal amounts ofenantiomers of a chiral molecule.

Diastereomer

The term “diastereomer” as used herein means one of a class ofstereoisomers that are not enantiomers, but that have differentconfigurations at one or more of the equivalent chiral centers. Exampleof diasteromers are epimers that differ in configuration of only onechiral center.

Stereoisomer

The term “stereoisomer” as used herein means one of a class of isomericmolecules that have the same molecular formula and sequence of bondedatoms, but different three-dimensional orientations of their atoms inspace.

Prodrug

A prodrug is a medication that is administered as an inactive (or lessthan fully active) chemical derivative that is subsequently converted toan active pharmacological agent in the body, often through normalmetabolic processes.

Independently

Where two or more moieties are described as being “each independently”selected from a list of atoms or groups, this means that the moietiesmay be the same or different. The identity of each moiety is thereforeindependent of the identities of the one or more other moieties.

Embodiments of the present invention are described below. Preferredfeatures of each aspect of the present invention are as for each of theother aspects mutatis mutandis. Moreover, it will be appreciated thatthe features specified in each embodiment may be combined with otherspecified features, to provide further embodiments.

Among the several established animal models, repetitive intraperitoneal(i.p.) injection of cholecystokinin secretagogue, cerulein, is the mostwidely used and a highly reproducible method for the production of anexperimental acute pancreatitis. Followed by a single shot oflipopolysaccharide (LPS), pulmonary injury characterized by neutrophilsequestration in the lung tissues and increased permeability of thealveolar membrane barrier is often observed as an acute pancreatitisassociated complication. For the diagnosis of the onset of acutepancreatitis, bulky leakage of digestive enzymes, namely α-amylase, intothe bloodstream is regarded as the principal pathological parameter. Forevaluating the severity of acute pancreatitis and the associatedpulmonary injury, morphological alterations of organ architectureincluding interstitial edema, cellular damage, leukocyte infiltrationand hemorrhage are characterized as the histological and/or pathologicalparameters. Besides histological examination, aberrant MPO activity isoften measured for assessing the severity of neutrophil-mediatedinflammatory condition. Both the local and systemic inflammatoryresponses can be further confirmed by the high levels ofpro-inflammatory cytokines present in the homogenates of affectedtissues. Moreover, glutathione depletion, a defense mechanism, is one ofthe most common parameters for assessing the severity of tissue injury.

The subject to be treated by the method of this invention may be a humanor an animal. The present invention is applicable to various forms ofacute pancreatitis, and particularly to the acute pancreatitisassociated systemic complications including pulmonary injury.

Dihydro-resveratrol, also known as trans-3,5,4′-trihydroxybibenzyl, is aderivative of polyphenol belonging to the family of stilbenoids, whichare often obtained from plant extracts. In fact, dihydro-resveratrol isa phytoalexin produced by various plant species including Orchidaceaeand Cannabis sativa L. against abiotic and biotic challenges,particularly in the case of fungal infection as reported in Fritzemeier,K. H., Kindl, H. 1983. 9,10-dihydrophenanthrenes as phytoalexins ofOrchidaceae. Biosynthetic studies in vitro and in vivo proving the routefrom L-phenylalanine to dihydro-m-coumaric acid, dihydrostilbene anddihydrophenanthrenes. Eur J Biochem 133, 545-550.

The present invention relates to the usage of a polyphenol derivative ofthe stilbenoid family with formula 1:

wherein R₁, R₂ and R₃ are each independently selected from an alkylgroup. The term “alkyl”, alone or in combination with other groups,includes reference to a straight chain alkyl moiety having 1, 2, 3, 4, 5or 6 carbon atoms. The term is further exemplified by such groups asmethyl, ethyl, propyl (n-propyl or isopropyl), butyl (n-butyl, sec-butyland tert-butyl), pentyl, hexyl and the like, to ameliorate tissue injuryof the pancreas and lungs.

The present invention further relates to the usage of a stilbenecompound containing trans-3,5,4′-trihydroxybibenzyl, also known asdihydro-resveratrol, see Compound 2:

to ameliorate tissue injury of the pancreas and lungs. In the presentinvention, this particular stilbenoid derivative was obtained as whitepowders through hydrogenation of trans-resveratrol.

Further, the invention is concerned with a process for the manufactureof the above compound, pharmaceutical preparations which contain suchcompound, and the use of this compound for the production ofpharmaceutical preparations.

The oral administration of dihydro-resveratrol at an adequate dosage ofnot less than 20 mg/kg is shown to significantly ameliorate the severityof acute pancreatitis and associated pulmonary injury incerulein-treated rats. In terms of pathological parameters, rats withacute pancreatitis are shown to have lessened pancreatic water contentdue to an attenuation of pancreatic edema (FIG. 1), lowered plasma levelof α-amylase (Table 1), more intact acinar morphology (FIGS. 2C to 2F)and reduced thickening of alveolar wall and hemorrhage (FIGS. 3C to 3E).

TABLE 1 Plasma α-amylase activities are expressed as U/μl/minute. Ap-value of less than 0.05 is considered as statistically significant andS.D. stands for standard derivation. Cerulein + Cerulein + Cerulein +D-Res D-Res D-Res Control Cerulein 10 mg 20 mg 50 mg mean 0.1294  0.4846 * 0.2891  0.2498 #  0.2431 # S.D. 0.03909 0.1457 0.052480.05593 0.06025 * p < 0.05 when comparing with control group whereas # p< 0.05 comparing with cerulein group.

Cerulein-induced elevated levels of neutrophil sequestration, which isquantified as the activity of MPO, are significantly suppressed inpancreatic and pulmonary tissues by the administration ofdihydro-resveratrol (FIGS. 4A and 4B). Cerulein-induced elevated levelsof TNF-α in the pancreas and lungs are significantly suppressed by theadministration of dihydro-resveratrol (FIGS. 5A and 5B).

Glutathione depletion is a distinctive sign of tissue injury. Thecerulein-induced declined levels of glutathione in the pancreas aresignificantly restored by the administration of dihydro-resveratrol(FIG. 6A).

In the present invention, dihydro-resveratrol completely and easilydissolves in 0.5% (weight/volume, w/v) methanol whereastrans-resveratrol, with vigorous shaking, dissolves in 2.5% (w/v)methanol (Table 2). Thus, the solubility of dihydro-resveratrol is atleast 5 times higher than that of trans-resveratrol. The ameliorativeeffect of dihydro-resveratrol was more promising than that oftrans-resveratrol on reducing water content as a result of pancreaticedema in rats with cerulein-induced acute pancreatitis (FIG. 6B).

TABLE 2 Solubility of Dihydro-resveratrol and Trans-resveratrol inmethanol. Trans-resveratrol Dihydro-resveratrol Methanol required (w/v)2.5% 0.5%

From the evaluation of mitochondrial metabolic rates by means of MTTassay, the cytotoxicity of dihydro-resveratrol in pancreatic acinarcells is determined to be approximately 500 μM whereas that oftrans-resveratrol is roughly 250 μM (FIG. 7). Thus, the cytotoxicity ofdihydro-resveratrol was 50% lower than that of trans-resveratrol.

Experiments

Preparation and structural identification of dihydro-resveratrol. Themolecular formula of dihydro-resveratrol was established as C₁₄H₁₄O₃,which was obtained as white powders through hydrogenation oftrans-resveratrol. A solution of trans-resveratrol (10 g, 43.8 mmol) inanhydrous EtOH (150 ml) was stirred at room temperature under 5 atm H₂pressure in the presence of 10% Pd/C (0.2 g). The reaction was quenchedafter 8 hours (h), by filtering off the catalyst. The filtrate wasevaporated in vacuum and the residue was subjected to silica gelchromatographic separation eluting with petroleum ether and ethylacetate (1:1) to afford dihydro-resveratrol as white amorphous powder(9.6 g, 95% yield): HR-ESIMS ([M+1]+ m/z 231.1026, calcd 231.1016 forC₁₄H₁₅O₃); ¹H NMR (methanol-d4, 400 MHz) δ 6.96 (2H, ABd, J=8.3 Hz),6.67 (2H, ABd, J=8.4 Hz), 6.13 (2H, brd, J=2.2 Hz), 6.09 (1H, brt, J=2.2Hz), 2.74 (2H, brdd, J=8.5, 5.6), 2.67 (2H, brdd, J=8.3, 5.2); ¹³C NMR(methanol-d4, 100 MHz) δ 159.2 (2C, s), 156.3 (1C, s), 145.6 (1C, s),134.1 (1C, s), 130.3 (2C, d), 116.0 (2C, d), 108.1 (2C, d), 101.1 (1C,d), 39.6 (2C, t), 38.0 (2C, t).

Evaluation of biological activities. Sprague-Dawley rats aged 28 daysweighing in the range of 70 to 90 g were randomly assigned into 6 groupsof 6 to 8 individuals. The rats were housed with an ambient temperatureof 23±2° C., a relative humidity of 60 to 80% and a 12-h light/darkcycle. Prior to the experiment, the rats were starved overnight butallowed with free access to water. Experimental acute pancreatitis wasinduced in the rats by six hourly i.p. injections of cerulein at thesupramaximally stimulating dose (50 μg/kg) followed by a single dose ofLPS at 7.5 mg/kg 1 h after the last cerulein injection, and this groupof rats was designated as the cerulein group. The control group receivedinjections of 0.9% saline instead of cerulein in the same volume and atsame time intervals. The treatment groups given with cerulein and oraldoses of dihydro-resveratrol (10, 20 or 50 mg/kg) were designated asCerulein+D-res 10 or 20 or 50 mg/kg. The therapeutic intervention wasgiven 30 minutes after the first cerulein injection for threeconsecutive hours. Upon scarification, pancreata were immediatelyremoved, weighed, trimmed from fat and fixed in 4%paraformaldehyde-phosphate buffered saline overnight at 4° C. Sampleswere then processed, embedded in paraffin wax, sectioned and subjectedto H&E staining. Levels of TNF-α in pancreatic and pulmonary sampleswere determined using commercial ELISA kits. Tissue homogenates weresubjected to biochemical assays for the evaluation of MPO activity andglutathione content.

Functional intact acini were dissociated from pancreatic tissue usingcollagenase digestion with mild shearing forces. Acini were cultured inDulbecco's modification of Eagle's medium (GIBCO) supplemented with 5%fetal bovine serum (GIBCO), 1% penicillin-streptomycin (GIBCO) in a 5%CO₂, 95% air humidified atmosphere at 37° C. LTC-14 cells were seeded ata density of 1×10⁴/well in a 96-well plate, and incubated with differentconcentrations of dihydro-resveratrol or trans-resveratrol (dissolved inDMSO) for 24 hours. MTT reagent was added to the cells at the end of the24-hour treatment period. After a 3-hour reaction time, MTT productswere dissolved in DMSO and absorbance at 570 nm was taken.

Results

After the induction of cerulein, the weight ratio of pancreas to body inthe acute pancreatitis rats was drastically increased by roughly 60%when compared with the non-cerulein induced controls due to theoccurrence of pancreatic edema. The oral administration ofdihydro-resveratrol at an adequate dosage of not less than 20 mg/kgnotably reduced the pancreatic edema as reflected by the significantdecrease in the weight ratio of pancreas to body. The ameliorativeeffect of dihydro-resveratrol on reducing pancreatic edema was morepromising than that of trans-resveratrol, the accredited antioxidant.Regarding the human dosage, the comparable dosage is 3.24 mg/kg based onthe standard dosage conversion according to Reagan-Shaw S, Nihal M,Ahmad N (2008) Dose translation from animal to human studies revisited.FASEB J 22(3): 659-661.

When oral administration of dihydro-resveratrol was given, the focalexpansion of the interlobular septae, cytoplasmic shrinkage andleukocyte infiltration in pancreatitic parenchyma was remarkably reducedwhereas the pulmonary wall thickening and hemorrhage in lung tissueswere significantly improved in the rats with cerulein-induced acutepancreatitis.

For a relief of inflammatory conditions of the pancreas and lungs, thelevels of pro-inflammatory cytokine TNF-α as well as MPO activities weresignificantly reduced in the pancreatic and pulmonary tissues by theoral administration of dihydro-resveratrol.

The level of glutathione in cerulein-induced pancreatic tissue wasdepleted drastically by more than 50% when compared to thenon-cerulein-treated control. The oral administration ofdihydro-resveratrol significantly suppressed glutathione depletion inthe cerulein-induced pancreata.

The solubility of dihydro-resveratrol in a methanol-based solvent was atleast 5 times higher than that of trans-resveratrol. By assessing themitochondrial metabolic rates of acini, the cytotoxicity ofdihydro-resveratrol was shown to be approximately 500 μM whereas that oftrans-resveratrol was roughly 250 μM. Thus, the cytotoxicity ofdihydro-resveratrol was 50% lower than that of trans-resveratrol.

In a first embodiment of a first aspect of the present invention thereis provided a method of treating acute inflammatory condition of thepancreas and associated systemic complications by administering to asubject in needs thereof a composition comprising an effective amount ofa stilbenoid derivative which comprises a compound of formula (1),

wherein R₁, R₂ and R₃ are each independently selected from an alkylgroup. The term “alkyl”, alone or in combination with other groups,includes reference to a straight chain alkyl moiety having 1, 2, 3, 4, 5or 6 carbon atoms. The term is further exemplified by such groups asmethyl, ethyl, propyl (n-propyl or isopropyl), butyl (n-butyl, sec-butyland tert-butyl), pentyl, hexyl and the like, and the derivatives orchemical variants thereof; or a mixture of said compound, the derivativeand/or chemical variants thereof.

In a second embodiment of a first aspect of the present invention thereis provided a method of treating acute inflammatory condition of thepancreas and associated systemic complications wherein the stilbenoidderivative is trans-3,5,4′-trihydroxybibenzyl, or dihydro-resveratrol,which is a compound of formula (2):

and the derivatives or chemical variants thereof; or a mixture of saidcompound, the derivative and/or chemical variants thereof.

In a third embodiment of the first aspect of the present invention thereis provided a method of treating acute inflammatory condition of thepancreas and associated systemic complications wherein the subject is ahuman or an animal.

In a fourth embodiment of the first aspect of the present inventionthere is provided a method of treating acute inflammatory condition ofthe pancreas and associated systemic complications wherein thecomposition is administered orally.

In a fifth embodiment of the first aspect of the present invention thereis provided a method of treating acute inflammatory condition of thepancreas and associated systemic complications wherein the acuteinflammatory condition of the pancreas comprises all forms of acutepancreatitis and associated systemic complications comprise pulmonaryinjury.

In a sixth embodiment of the first aspect of the present invention thereis provided a method of treating acute inflammatory condition of thepancreas and associated systemic complications wherein said compositionis administered at no less than 20 mg/kg to said subject for no lessthan 3 times a day.

In a seventh embodiment of the first aspect of the present inventionthere is provided a method of treating acute inflammatory condition ofthe pancreas and associated systemic complications wherein saidcomposition is administered at no less than 3.24 mg/kg to said subjectfor no less than 3 times a day.

In a first embodiment of a second aspect of the present invention thereis provided a method for preparing a compound of molecular formulaC₁₄H₁₄O₃ and of formula (2),

which is a stilbenoid derivative having a chemical name oftrans-3,5,4′-trihydroxybibenzyl by hydrogenating of trans-resveratrol.

In a second embodiment of the second aspect of the present inventionthere is provided a method of preparing the compound of molecularformula C₁₄H₁₄O₃ and of formula (2) wherein the hydrogenating oftrans-resveratrol comprises steps of

-   -   stirring a solution of trans-resveratrol in anhydrous EtOH at        room temperature under 5 atm H₂ pressure in the presence of 10%        Pd/C for 8 hours;    -   filtering off the catalyst from the stirred solution;    -   evaporating the filtrate in vacuum to produce a residue;    -   eluting the residue using silica gel chromatographic separation        with petroleum ether and ethyl acetate (1:1) to produce        dihydro-resveratrol.

Further Embodiments of the Present Invention

TGF-β has been reported by some previous studies as a potent inducer ofPSC activation in which a series of fibrotic mediators, including FN1,are being up-regulated. In cultured LTC-14 cells, which are immortalizedPSCs from rat, the expression levels of fibrotic filament α-SMA and ECMprotein FN1 are remarkably elevated by the exogenous addition ofrecombinant TGF-β (5 ng/mL). In one further embodiment of the presentinvention, the inventors discover that the administration ofdihydro-resveratrol significantly attenuate the expression levels ofα-SMA and FN1 in rat PSCs upon the challenge of TGF-β. The derivativesof dihydro-resveratrol exert similar suppressive effect in PSCs. Whencompared to the renowned anti-oxidant trans-resveratrol, the inhibitoryeffect of dihydro-resveratrol is more significant. Among the testingstilbenoids, dihydro-resveratrol exerts the most potent anti-fibroticeffect in PSCs despite they possess modest structural differences.

The present invention provides a compound for suppressing a fibroticmediator of stellate cells present in an internal organ of a subject inneed with a formula of

wherein R² and R⁴ are each independently selected from —OR¹¹ and—OC(O)R¹;

R¹, R³, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are each independently selected fromhydrogen, halogen, trifluoromethyl, —OR¹¹ and —OC(O)R¹¹; or R² and R³,or R⁷ and R⁸ may be taken together with the carbon atoms to which theyare attached to form a cyclic group;

R¹¹ is independently hydrogen or selected from hydrocarbyl andheterocyclyl, either of which is optionally substituted with 1, 2, 3, 4or 5 R¹²;

R¹² is independently selected from halogen, trifluoromethyl, cyano,nitro, oxo, —OR¹³, —C(O)R¹⁴, —C(O)N(R¹³)R¹⁴, —C(O)OR¹³, —OC(O)R¹⁴,—S(O)₂R¹³, —S(O)₂N(R¹³)R¹⁴, —N(R¹³)R¹⁴;

R¹³ and R¹⁴ are each independently hydrogen or selected from hydrocarbyland heterocyclyl, either of which is optionally substituted with 1, 2,3, 4 or 5 substituents independently selected from halogen, cyano,amino, hydroxy, C₁₋₆ alkyl and C₁₋₆ alkoxy;

or an enantiomer thereof;

or a pharmaceutically acceptable salt or prodrug thereof,

or a mixture of said compound, the derivative and/or chemical variantsthereof.

The present invention further provides nine embodiments of compounds forsuppressing a fibrotic mediator of stellate cells present in an internalorgan of a subject in need with formula of:

The internal organ can be, for example, pancreas, liver, kidney and lungof a subject. The subject can be a human subject.

Experiments

LTC-14 cells were cultured at 37° C. under a humidified condition of 95%air and 5% CO₂ in IMDM supplemented with 10% fetal bovine serum (FBS).Cells used in all the experiment were among passages 9 to 25. LTC-14cells were seeded at a density of 1×10⁵/well in a 12-well plate, andincubated with recombinant TGF-β at 5 ng/mL with dihydro-resveratrol at0, 1, 5, 10 and 20 μg/mL in IMDM supplemented with 0.2% FBS for 24hours. Cells were then harvested for protein extraction and Westernblotting analysis or immunofluorescent staining.

Total proteins of the LTC-14 cells are extracted using RIPA lysisbuffers containing protease inhibitors. Cell lysates were loaded andseparated by SDS-polyacrylaminde gel electrophoresis. After wetelectroblotting, proteins were transferred onto PVDF membranes(Bio-rad), blocked with 5% non-fat milk, probed with antibodies andvisualized by utilization of an ECL kit (GE Healthcare).

For immunofluorescent staining of α-SMA, LTC-14 cells were seeded at adensity of 1×10⁵ onto the poly-L-lysine-coated cover slips in a 24-wellplate, incubated with TGF-β at 5 ng/mL with dihydro-resveratrol at 0,and 10 g/mL in IMDM supplemented with 0.2% FBS for 24 hours. Cells werethen fixed, blocked with 3% BSA, probed with antibodies and mounted withfluorescence mounting medium containing 4′, 6-diamidino-2-phenylindole(DAPI). Images were captured using the Nikon microscope and analyzed bythe SPOT advanced software.

Evaluation of biological activities. C57/BL6 mice aged 28 days weighingin the range of 20 to 25 g were randomly assigned into 4 groups of 6 to8 individuals. The mice were housed with an ambient temperature of 23±2°C., a relative humidity of 60 to 80% and a 12-h light/dark cycle. Priorto the glucose tolerance test, the mice were starved overnight butallowed with free access to water. Experimental chronic pancreatitis wasinduced in the mice by four hourly intraperitoneal injections ofcerulein at the supramaximally stimulating dose (50 μg/kg/h) a day, 3days a week, in a total of 6 weeks. The control group receivedinjections of 0.9% saline instead of cerulein in the same volume and atsame time intervals. The treatment groups given with cerulein and oraldoses of dihydro-resveratrol (20 mg/kg/day) were designated asCer+D-res. A dosage of dihydro-resveratrol at 50 mg/kg/day had also beenattempted in the treatment course, but no statistically significantdifference from the dose at 20 mg/kg/day in fibrosis formation wasachieved. Nevertheless, no adverse effect was obtained from this higherdosage in the in vivo trial. Thus, it concludes that an effective dosageof dihydro-resveratrol is at least 20 mg/kg/day. The group given withtrans-resveratrol was designated as Cer+Res. The drug intervention ofboth compounds was given from the first day of week 4 till the end ofexperiment, i.e. in a total of 3 weeks. At the end of the 6-weekexperiment, mice were subjected to the intraperitoneal glucose tolerancetest (IPGTT). Mice had been starved for 14 hours prior to the IPGTT, inwhich a 15% (w/v) glucose solution was injected to individual animals at1.5 g glucose per kg body weight. About 1 μL of blood will be obtainedfrom the tail vein, and blood glucose levels were monitored using aglucometer (Medisign, Korea) 30 min before (i.e. fasting level) and 10,20, 30 and 60 min after glucose injection. Upon scarification,pancreases were immediately removed, weighed, trimmed from fat and fixedin 4% paraformaldehyde-phosphate buffered saline overnight at 4° C.Samples were then processed, embedded in paraffin wax, sectioned andsubjected to immunostaining.

According to the dose translation formula, human equivalent dosage(mg/kg)=Animal dose (mg/kg) multiplied by animal Km/human Km, wheremouse Km is 3 and human Km is 37 (Guidance for Industry Estimating theMaximum Safe Starting Dose in Initial Clinical Trials for Therapeuticsin Adult Healthy Volunteers), the effective human equivalent dosage ofdihydro-resveratrol of the present invention is at least 1.622mg/kg/day.

In yet another embodiment of the present invention, 8 other derivatives(compounds i to viii) of dihydro-resveratrol and (compound 2)dihydro-resveratrol are shown in FIGS. 8A-81. In this embodiment, eachcompound is experimented accordingly as follows:

Experiment with the Embodiments of Compounds in FIGS. 8A-81

LTC-14 cells were pre-incubated with TGF-β (5 ng/mL), and treated withtrans-resveratrol (Resv) or dihydro-resveratrol (D-Res) or stilbenecompounds i-viii at 20 μg/mL for 24 hours. Control was not treated withResv or any stilbenoids. Total proteins were extracted and analyzedusing Western blotting. This is shown in FIG. 9.

LTC-14 cells are pancreatic stellate cells. α-SMA is the hallmarkcomponent of fibrogenesis whereas β-actin serves as a loading control.Thus, the expression level of α-SMA implies the degree of PSCactivation. TGF-β was added since it is regarded as a potent inducer offibrotic events. Suppressive effect on α-SMA expression level is testedamong dihydro-resveratrol and compounds i to viii in relation totrans-resveratrol (Resv). All of the testing compounds exert suppressiveeffect of α-SMA expression level.

LTC-14 cells were pre-incubated with TGF-β (5 ng/mL), and treated withtrans-resveratrol or dihydro-resveratrol at the indicatedconcentrations. Total proteins were extracted and analyzed using Westernblotting. This is shown in FIG. 10.

FN1 is a major extracellular matrix protein produced during fibrogenesisor upon the activation of pancreatic stellate cells. Its expressionlevel implies the degree of fibrogenesis. Suppressive effect on levelsof FN1 and α-SMA is tested between dihydro-resveratrol (i.e. compound 2)and trans-resveratrol.

LTC-14 cells were pre-incubated with TGF-β (5 ng/mL), and treated withdihydro-resveratrol at 20 μg/mL for 24 hours prior to immunofluorescentstaining.

LTC-14 cells are pancreatic stellate cells. α-SMA is the hallmarkcomponent of fibrogenesis whereas β-actin serves as a loading control.Thus, the green fluorescent signal (identified by arrow marks in FIG.11) of α-SMA implies the degree of PSC activation. TGF-β was added sinceit is regarded as a potent inducer of fibrotic events. Suppressiveeffect of dihydro-resveratrol on α-SMA expression level is tested.

Pancreatic tissue sections are stained with antibody against FN1; thus,the immunostaining signals imply the degree of FN1 deposition in theparenchyma. The treatment with dihydro-resveratrol at 20 mg/kg/day(Cer+D-Res) reduces such deposition in chronic pancreatitis in a mannermore significant to trans-resveratrol (Cer+Res). This is shown in FIG.12.

Evaluation of biological activities. C57/BL6 mice aged 28 days weighingin the range of 20 to 25 g were randomly assigned into 4 groups of 6 to8 individuals. The mice were housed with an ambient temperature of 23±2°C., a relative humidity of 60 to 80% and a 12-h light/dark cycle. Whenoral administration of dihydro-resveratrol (20 mg/kg/day) is given, thefasting blood glucose levels of the chronic pancreatitis mice (Cer)become markedly higher than those of the control group, indicating thesechronic pancreatitis mice develop hyperglycemia, a discernible featureof diabetes. Importantly, their impaired glucose tolerance has beensignificantly rectified by the 3-week dihydro-resveratrol treatment(Cer+D-Res). As a result, the hyperglycemic condition of the chronicpancreatitis mice is improved. This is shown in FIG. 13.

When oral administration of dihydro-resveratrol (Cer+D-Res) is given at20 mg/kg/day, the severity of pancreatitis and the shrinkage anddestruction of islets, explicitly beta cells, are notably ameliorated.As shown in FIG. 14, the pancreatic beta-cell area is reflected by theimmunofluorescent insulin signals. A reduced beta-cell area or massimplicates the deficiency in glucose tolerance, or the development ofdiabetes. Thus, the restoration of beta-cell area by thedihydro-resveratol treatment indicates this stilbenoid is beneficial tothe treatment of pancreatogenic diabetes (i.e. Type3c DM).

According to the dose translation formula, human equivalent dosage(mg/kg)=Animal dose (mg/kg) multiplied by animal Km/human Km, wheremouse Km is 3 and human Km is 37 (Guidance for Industry Estimating theMaximum Safe Starting Dose in Initial Clinical Trials for Therapeuticsin Adult Healthy Volunteers), the effective human equivalent dosage ofdihydro-resveratrol of the present invention is at least 1.622mg/kg/day.

INDUSTRIAL APPLICABILITY

The present invention relates to a polyphenol derivative of thestilbenoid family, namely trans-3,5,4′-trihydroxybibenzyl, also known asdihydro-resveratrol, as a remedial agent. In particular, the presentinvention presents the usage of dihydro-resveratrol as an anti-fibroticagent in suppressing the activation of pancreatic stellate cells (PSCs).One embodiment of the present invention also relates to the managementof pancreatic fibrosis, which is often accompanied with chronicpancreatitis and desmoplastic reaction of pancreatic cancer. Anotherembodiment of the present invention also has applications in preventingor alleviating or treating pancreatic tumors or pancreatic tumorsrelated diseases or pancreatogenic diabetes.

1. A compound for suppressing a fibrotic mediator of stellate cellspresent in an internal organ of a subject in need with a formula of

wherein R² and R⁴ are each independently selected from —OR¹¹ and—OC(O)R¹¹; R¹, R³, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are each independentlyselected from hydrogen, halogen, trifluoromethyl, —OR¹¹ and —OC(O)R¹¹;or R² and R³, or R⁷ and R⁸ may be taken together with the carbon atomsto which they are attached to form a cyclic group; R¹¹ is independentlyhydrogen or selected from hydrocarbyl and heterocyclyl, either of whichis optionally substituted with 1, 2, 3, 4 or 5 R¹²; R¹² is independentlyselected from halogen, trifluoromethyl, cyano, nitro, oxo, —OR¹³,—C(O)R¹⁴, —C(O)N(R¹³)R¹⁴, —C(O)OR¹³, —OC(O)R¹⁴, —S(O)₂R¹³,—S(O)₂N(R¹³)R¹⁴, —N(R¹³)R¹⁴; R¹³ and R¹⁴ are each independently hydrogenor selected from hydrocarbyl and heterocyclyl, either of which isoptionally substituted with 1, 2, 3, 4 or 5 substituents independentlyselected from halogen, cyano, amino, hydroxy, C₁₋₆ alkyl and C₁₋₆alkoxy; or an enantiomer thereof; or a pharmaceutically acceptable saltor prodrug thereof; or a mixture of said compound, the derivative and/orchemical variants thereof.
 2. A compound for suppressing a fibroticmediator of stellate cells present in an internal organ of a subject inneed with a formula of


3. A method for suppressing fibrotic mediator(s) in stellate cellspresent in an internal organ of a subject in need by using a compositioncomprising compounds according to claim
 1. 4. The method according toclaim 3 wherein the composition is administered to a subject in needthereof with a dosage of at least 1.622 mg/kg/day wherein saidcomposition is consist of a compound with a formula of


5. The method according to claim 4 wherein the said subject is human. 6.The method according to claim 4 wherein the composition is administeredorally to said subject in need thereof.
 7. The method according to claim4 wherein the internal organ comprises pancreas, liver, kidney and lung.8. A compound for treating pancreatogenic diabetes (or Type 3c diabetesmilieus) in a subject in need with a therapeutically effective amount ofa formula of

wherein R² and R⁴ are each independently selected from —OR¹¹ and—OC(O)R¹¹; R¹, R³, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are each independentlyselected from hydrogen, halogen, trifluoromethyl, —OR¹¹ and —OC(O)R¹¹;or R² and R³, or R⁷ and R⁸ may be taken together with the carbon atomsto which they are attached to form a cyclic group; R¹¹ is independentlyhydrogen or selected from hydrocarbyl and heterocyclyl, either of whichis optionally substituted with 1, 2, 3, 4 or 5 R¹²; R¹² is independentlyselected from halogen, trifluoromethyl, cyano, nitro, oxo, —OR¹³,—C(O)R¹⁴, —C(O)N(R¹³)R¹⁴, —C(O)OR¹³, —OC(O)R¹⁴, —S(O)₂R¹³,—S(O)₂N(R¹³)R¹⁴, —N(R¹³)R¹⁴; R¹³ and R¹⁴ are each independently hydrogenor selected from hydrocarbyl and heterocyclyl, either of which isoptionally substituted with 1, 2, 3, 4 or 5 substituents independentlyselected from halogen, cyano, amino, hydroxy, C₁₋₆ alkyl and C₁₋₆alkoxy; or an enantiomer thereof; or a pharmaceutically acceptable saltor prodrug thereof; or a mixture of said compound, the derivative and/orchemical variants thereof.
 9. A compound for treating pancreatogenicdiabetes (or Type 3c diabetes milieus) in a subject in need with atherapeutically effective amount of a formula of


10. A method for treating pancreatogenic diabetes (or Type 3c diabetesmilieus) in a subject in need by using a composition comprisingcompounds according to claim
 8. 11. The method according to claim 10wherein the composition is administered to a subject in need thereofwith a dosage of at least 1.622 mg/kg/day wherein said composition isconsist of a compound with a formula of


12. The method according to claim 10 wherein the said subject is human.13. The method according to claim 10 wherein the composition isadministered orally to said subject in need thereof.